Item Type: | Article |
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Title: | Gateway compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells |
Creators Name: | Petrakis, S., Rasko, T., Mates, L., Ivics, Z., Izsvak, Z., Kouzi-Koliakou, K. and Koliakos, G. |
Abstract: | The Gateway Technology cloning system and the transposon technology represent state-of-the-art laboratory techniques. Combination of these molecular tools may allow rapid cloning of target genes into expression vectors. Here, we describe a novel Gateway Technology compatible transposon plasmid that combines the advantages of the Gateway recombination cloning with the Sleeping Beauty (SB) transposon-mediated transgene integrations. In our system the transposition is catalyzed by the novel hyperactive SB100x transposase providing the high efficient and precise transgene integrations into the host genome. A Gateway compatible transposon plasmid was generated in which the potential target gene may be fused with a Yellow Fluorescent Protein (YFP) tag at the N'- terminal. The vector utilizes the CAGGS promoter to control the fusion protein expression. The transposon expression vector encoding fusion YFP-IFNB1 protein together with the hyperactive SB100x transposase was used to generate stable cell lines in human embryonic kidney (HEK293) and rat adipose-derived stromal cells (ASC). HEK293 and ASCs stably expressed and secreted human interferon-beta protein up to 4 weeks after transfection. The generated Gateway compatible transposon plasmid may be utilized for numerous experimental approaches, such as gene therapy or high-throughput screening methods in primary cells, representing a valuable molecular tool for laboratory applications. |
Keywords: | Methods, Gene Expression, Transposon, Gateway, Animals, Rats |
Source: | Biotechnology Journal |
ISSN: | 1860-6768 |
Publisher: | Wiley |
Volume: | 7 |
Number: | 7 |
Page Range: | 891-897 |
Date: | July 2012 |
Official Publication: | https://doi.org/10.1002/biot.201100471 |
PubMed: | View item in PubMed |
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